Acanthamoeba castellanii is one of several closely related species that cause the eye disease Acanthamoeba keratitis. Acanthamoeba can also harbor a wide variety of bacterial pathogens, for which it may serve as an environmental reservoir. Notwithstanding its pathogenicity, Acanthamoeba has served as a model organism for a wide range of biochemical studies, all of which have been hampered by the lack of a transfection system. A method for stable transfection of Acanthamoeba was recently developed, providing a useful tool for several investigations, but the first generation of transfection vectors have not been optimized with respect to copy number, structure within amoebae, levels of neomycin phosphotransferase expression and reporter gene expression. In order to develop a versatile system for expressing proteins in Acanthamoeba at defined levels it is proposed to examine the effects of promoter strength, the nature of 5' and 3' untranslated sequences and the presence of introns on the efficacy of neomycin phosphotransferase expression. Results will be evaluated on the basis of plasmid structure and copy number, efficiency of transfection and levels of neomycin phosphotransferase production. Stable integrants will be produced by homologous recombination, so that permanent cell lines can be established and gene function can be studied in a more physiological context. Similar approaches will be used to optimize reporter gene expression from the same plasmid but will incorporate synthetic and inducible promoters in order to provide control over the level of expression to range from physiological expression levels to very efficient promoters for recombinant protein production. Inducible promoters will permit controlled expression of either toxic proteins or RNAi against essential proteins. Tandem affinity tags will be used in order to permit investigations of protein-protein interactions and the purification of low-abundance multi-subunit complexes. Collectively, the optimized vectors will provide a versatile platform for the in vivo study of processes ranging from gene expression to pathogenicity. Acanthamoeba castellanii is an opportunistic human pathogen causing the severe eye disease Acanthamoeba keratitis and can cause brain infection and various infections of immunocompromised individuals. Acanthamoeba can harbor a wide range of bacterial and fungal pathogens, and growth of these pathogens within Acanthamoeba may select for virulence. This proposal will develop genetic techniques for the biological study of Acanthamoeba as well as its interactions with other pathogens. [unreadable] [unreadable] [unreadable]